N-Formyl Hydroxylamines

ABSTRACT

Novel N-formyl hydroxylamine compounds and their derivatives are disclosed. These N-formyl hydroxylamine compounds inhibit peptidyl deformylase (PDF), an enzyme present in prokaryotes. The compounds are useful as antimicrobials and antibiotics. The compounds of the invention display selective inhibition of peptidyl deformylase versus other metalloproteinases such as MMPs. Methods of preparation and use of the compounds are also disclosed.

This invention is directed to novel N-formyl hydroxylamine compounds, tothe uses of these compounds in various medicinal applications, includingtreating disorders amenable to treatment by peptidyl deformylaseinhibitors such as treatment of bacterial infections, and topharmaceutical compositions comprising these compounds.

Treatment of microbial infection in host organisms requires an effectivemeans to kill the microbe while doing as little harm to the host aspossible. Accordingly, agents which target characteristics unique to apathology-causing microorganism are desirable for treatment.

Peptide deformylase is a metallopeptidase found in prokaryotic organismssuch as bacteria. Protein synthesis in prokaryotic organisms begins withN-formyl methionine (fMet). After initiation of protein synthesis, theformyl group is removed by the enzyme peptide deformylase (PDF); thisactivity is essential for maturation of proteins.

Metalloproteinases are critical to many aspects of normal metabolism.Disorders involving metalloproteinases have been implicated in severaldiseases such as cancer, arthritis, and autoimmune diseases. Because ofthe importance of MMPs in normal physiological processes, it would bepreferable to develop agents that inhibit PDF while avoiding significantinhibition of MMPs. Alternatively, PDF inhibitors which also inhibitMMPs may be of use where the therapeutic benefits of inhibiting PDFoutweigh the risk of side effects from MMP inhibition.

Research on inhibitors of PDF is much less extensive than that forinhibitors of MMPs. N-formyl hydroxylamine derivatives are described inInternational Patent Application WO 99/39704 and WO 02/102790. In viewof the importance of identifying new antibiotics to treat bacteriaresistant to existing antibiotics, it is desirable to develop novelinhibitors of PDF for evaluation and use as antibacterial andantimicrobial agents. The present invention fulfills this need.

In particular, the present invention provides an N-formyl hydroxylaminederivatives referred to herein collectively as “compounds of theinvention”), a salt thereof or a prodrug thereof, e.g. a compound offormula (I):

wherein R₁ is hydrogen, alkyl, heteroalkyl, heterocycloalkyl, aryl orheteroaryl;R₃ is hydrogen, halogen, or alkoxy; andR₄ is aryl, or heteroaryl; orn is 0 to 3a salt thereof or a prodrug thereof.

In one aspect, R₄ is a heteroaryl of formula (II)

wherein each of R₆, R₇, R₈ and R₉ independently is hydrogen, alkyl,substituted alkyl, phenyl, halogen, hydroxy or alkoxy,e.g. wherein

-   a.) R₆ and R₈ are hydrogen, R₉ is hydrogen or alkyl and R₇ is alkyl,    substituted alkyl or phenyl;-   b.) R₆, R₇ and R₉ are hydrogen and R₈ is halogen, alkyl or    substituted alkyl-   c.) R₇, R₈ and R₉ are hydrogen and R₆ is hydroxyl.

In a particularly useful aspect, the heteroaryl is of the formula (II.1)

wherein R₆, R₇ and R₉ are as defined above for formula (II) and R₈ ishalogen, e.g. fluoro.

In yet another aspect, R₄ is of formula (II.2)

wherein R₆, R₇ and R₈ are as defined above for formula (II) above

In still another aspect, R₄ is of formula (II.3)

wherein R₆, R₇ and R₈ are as defined above for formula (II)

In still another aspect, R₄ is of formula (II.4)

wherein R₆, R₇ and R₈ are as defined above for formula (II)

In still another aspect, R4 is of formula (II.5)

wherein R₆, R₇ and R₈ are as defined above for formula (II)

Unless otherwise stated, the following terms as used in thespecification have the following meaning.

The term “cycloalkane” or cycloalkyl” contains from 3- to 7-ring carbonatoms, and is, preferably cyclopropyl, cyclobutyl, cyclopentyl, andcyclohexyl.

The term “aliphatic group” refers to saturated or unsaturated aliphaticgroups, such as alkyl, alkenyl or alkynyl, cycloalkyl or substitutedalkyl including straight-chain, branched-chain and cyclic groups havingfrom 1-10 carbons atoms. The term “alkyl” or “alk”, whenever it occurs,is a saturated straight chain or branched aliphatic group of 1-10 carbonatoms or a cycloalkyl of 3-10 carbon atoms, more preferably, alkylgroups are C₁-C₇alkyl, particularly, C₁-C₄alkyl. Examples of “alkyl” or“alk” include, but are not limited to, methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, neopentyl,n-hexyl or n-heptyl, cyclopropyl and, especially, n-butyl.

The term “substituted alkyl” refers to an alkyl group that issubstituted with one or more substitutents preferably 1 to 3substitutents including, but not limited to substituents such ashalogen, lower alkoxy, hydroxy, mercapto, carboxy, cycloalkyl, aryl,heteroaryl, and the like. Examples of substituted alkyl groups include,but are not limited to, —CF₃, —CF₂—CF₃, hydroxymethyl, 1- or2-hydroxyethyl, methoxymethyl, 1- or 2-ethoxyethyl, carboxymethyl, 1- or2-carboxyethyl, and the like.

The term “aryl” or “Ar” refers to an aromatic carbocyclic group of 6 to14 carbon atoms having a single ring (including, but not limited to,groups such as phenyl) or multiple condensed rings (including, but notlimited to, groups such as naphthyl or anthryl), and is especiallyphenyl.

The term “heteroaryl” or “HetAr” refers to a 4- to 7-membered,monocyclic aromatic heterocycle or a bicycle that is composed of a 4- to7-membered, monocyclic aromatic heterocycle and a fused-on benzene ring.The heteroaryl has at least one hetero atom, preferably at least twoheteroatoms including, but not limited to, heteroatoms such as N, O andS, within the ring. A preferred heteroaryl moiety is a 6 membered,monocyclic heterocycle having 2, 3 or 4 nitrogen heteroatoms in thering. Examples of heteteroaryl groups are pyridinyl, pyrimidinyl,pyrazinyl, pyridazinyl, pyridazinyl N-oxide or benzdioxolanyl, triazineor tetrazines.

The aryl or heteroaryl may be unsubstituted or substituted by one ormore substituents including, but not limited to C₁₋₇ alkyl, particularlyC₁₋₄ alkyl such as methyl, hydroxy, alkoxy, acyl, acyloxy, SCN, cyano,nitro, thioalkoxy, phenyl, heteroalkylaryl, alkylsulfonyl, halogen, andformyl.

The term “heteroalkyl” refers to saturated or unsaturated C₁₋₈ alkyl asdefined above, and especially C₁₋₄ heteroalkyl which contain one or moreheteroatoms, as part of the main, branched, or cyclic chains in thegroup. Heteroatoms may independently be selected from the groupconsisting of —NR— where R is hydrogen or alkyl, —S—, —O—, and —P—;preferably —NR— where R is hydrogen or alkyl, and/or —O—. Heteroalkylgroups may be attached to the remainder of the molecule either at aheteroatom (if a valence is available) or at a carbon atom. Examples ofheteroalkyl groups include, but are not limited to, groups such as—O—CH₃, —CH₂—O—CH₃, —CH₂—CH₂—O—CH₃, —S—CH₂—CH₂—CH₃, —CH₂—CH(CH₃)—S—CH₃,and —CH₂—CH₂—NH—CH₂—CH₂—.

The heteroalkyl group may be unsubstituted or substituted with one ormore substituents, preferably one to three substituents, including butnot limited to, alkyl, halogen, alkoxy, hydroxyl, mercapto, carboxy, andespecially phenyl. The heteroatom(s) as well as the carbon atoms of thegroup may be substituted. The heteroatom(s) may also be in oxidizedform.

The term “alkoxy” as used herein refers to a C₁₋₁₀ alkyl linked to anoxygen atom, or preferably C₁₋₇ alkoxy, more preferably C₁₋₄ alkoxy.Examples of alkoxy groups include, but are not limited to, groups suchas methoxy, ethoxy, n-butoxy, tert-butoxy, and allyloxy.

The term “halogen” or “halo” as used herein refer to chlorine, bromine,fluorine, iodine, and is especially fluorine.

“Protecting group” refers to a chemical group that exhibits thefollowing characteristics: 1) reacts selectively with the desiredfunctionality in good yield to give a protected substrate that is stableto the projected reactions for which protection is desired; 2) isselectively removable from the protected substrate to yield the desiredfunctionality; and 3) is removable in good yield by reagents compatiblewith the other functional group(s) present or generated in suchprojected reactions. Examples of suitable protecting groups may be foundin Greene et al., “Protective Groups in Organic Synthesis”, 2nd Ed.,John Wiley & Sons, Inc., New York (1991). Preferred amino protectinggroups include, but are not limited to, benzyloxycarbonyl (CBz),t-butyl-oxycarbonyl (Boc), t-butyldimethylsilyl(TBDMS),9-fluorenylmethyl-oxycarbonyl (Fmoc), or suitable photolabileprotecting groups such as 6-nitroveratryloxy carbonyl (Nvoc),nitropiperonyl, pyrenylmethoxycarbonyl, nitrobenzyl, dimethyldimethoxybenzyl, 5-bromo-7-nitroindolinyl, and the like. Preferredhydroxy protecting groups include Fmoc, TBDMS, photolabile protectinggroups (such as nitroveratryl oxymethyl ether (Nvom)), Mom (methoxymethyl ether), and Mem (methoxy ethoxy methyl ether). Particularlypreferred protecting groups include NPEOC (4-nitrophenethyloxycarbonyl)and NPEOM (4-nitrophenethyloxy-methyloxycarbonyl).

It will be appreciated that the compounds of formula (I) may exist inthe form of optical isomers, racemates or diastereoisomers. For example,a compound of formula (I) wherein R3 may be in the R- orS-configuration. It is to be understood that the present inventionembraces all enantiomers and their mixtures. Similar considerationsapply in relation to starting materials exhibiting asymmetric carbonatoms as mentioned.

The compounds of the invention may exist in the form of solidcrystalline salts. Preferably the crystalline salts are metal salts,preferably of divalent metals, although for some compounds it ispossible to form crystalline solids by using monovalent counter ions,such as Na. The counter ion is preferably Mg, Ca or Zn.

The compounds of the invention may typically be in the form of a hydrateor a mixed solvate/hydrate. Typically, the crystalline salt of theinvention contains about 2 to 8 waters of hydration, more typicallyabout 2 to 6 waters of hydration, and even more typically about 2 to 4waters of hydration. Thus, the crystalline salt of the inventiontypically comprises greater than 2% water, more typically about 4 toabout 12% water and even more typically about 8 to about 9% water.Solvates may be of one or more organic solvents, such as lower alkylalcohols, such as methanol, ethanol, isopropanol, butanol or mixturesthereof.

The compounds of the invention, e.g. the compounds of formula (I), mayexist in free form or in salt form, e.g. in form of a pharmaceuticallyacceptable salt. A “pharmaceutically acceptable salt” of a compoundmeans a physiologically and pharmaceutically acceptable salt thatpossesses the desired pharmacological activity of the parent compoundand does not impart undesired toxicological effects. Such salts include:

-   (1) acid addition salts, formed with inorganic acids such as    hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,    phosphoric acid, and the like; or formed with organic acids such as    acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic    acid, glycolic acid, pyruvic acid, lactic acid, malonic acid,    succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid,    citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid,    cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic    acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid,    benzenesulfonic acid, 4-chlorobenzenesulfonic acid,    2-napthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic    acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary    butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic    acid, hydroxynapthoic acid, salicylic acid, stearic acid, muconic    acid, and the like; or-   (2) salts formed when an acidic proton present in the parent    compound either is replaced by a metal ion, e.g. an alkali metal    ion, an alkaline earth ion, or an aluminum ion; or coordinates with    an organic base such as ethanolamine, diethanolamine,    triethanolamine, tromethamine, N-methylglucamine, and the like.

A compound of the invention, e.g. the compounds of formula (I), may actas a pro-drug. “Prodrug” means any compound which releases an activeparent drug according to formula (I) in vivo when such prodrug isadministered to a mammalian subject. Prodrugs of a compound of formula(I) are prepared by modifying functional groups present in the compoundof formula (I) in such a way that the modifications may be cleaved invivo to release the parent compound. Prodrugs include compounds offormula (I) wherein a hydroxy, amino, or sulfhydryl group is bonded toany group that may be cleaved in vivo to regenerate the free hydroxyl,amino, or sulfhydryl group, respectively. Examples of prodrugs include,but are not limited to esters (e.g. acetate, formate, and benzoatederivatives), carbamates (e.g. N,N-dimethylamino-carbonyl) of hydroxyfunctional groups in compounds of formula (I), and the like.

In the compounds of formula (I), the following significances arepreferred individually or in any sub-combination:

-   1. R₄ is a heteroaryl of formula of (II.1) wherein R₆, R₇ and R₉ are    hydrogen and R₈ is fluoro, R₆, R₇ and R₉ are hydrogen and R₈ is    methyl or trifluoromethyl; or R₆, R₇ and R₈ are hydrogen and R₉ is    fluoro; or R₆, R₈ and R₉ are hydrogen and R₇ is ethyl or methoxy; or    R₇, R₈ and R₉ are hydrogen and R₆ is hydroxy; or R₇ and R₈ are    hydrogen, R₆ is methoxy and R₉ is methyl; or R4 is a heteroaryl of    formula (II.2) wherein R₆, R₇ and R₈ are hydrogen, or R4 is a    heteroaryl of formula (II.3) wherein R6, R7 and R8 are hydrogen, or    R4 is a heteroaryl of formula (II.4) wherein R6, R7 and R8 are    hydrogen or R4 is a heteroaryl of formula (II.5) wherein R6, R7 and    R8 are hydrogen.-   2. R1 is alkyl, preferably n-butyl or cycloalkyl, preferably C₃₋₇    cycloalkyl such as cyclohexyl, cyclopropyl, or cyclopentyl-   4. R3 is halogen, preferably fluoro;

Utility

The compounds of the present invention can be used for the treatment orprevention of infectious disorders caused by a variety of bacterial orprokaryotic organisms. Examples include, but are not limited to, Grampositive and Gram negative aerobic and anaerobic bacteria, includingStaphylococci, e.g., S. aureus and S. epidermidis; Enterococci, e.g., E.faecalis and E. faecium; Streptococci, e.g., S. pneumoniae; Haemophilus,e.g., H. influenza; Moraxella, e.g., M. catarrhalis; and Escherichia,e.g., E. coli. Other examples include Mycobacteria, e.g., M.tuberculosis; intercellular microbes, e.g., Chlamydia and Rickettsiae;and Mycoplasma, e.g., M. pneumoniae; and Pseudomonas, e.g., P.aeruginosa; H. pylori; and parasites, e.g., Plasmodium falciparum.

Compounds of the present invention preferably have substantialimprovement in microbiological efficacy against either Gram positive orGram negative bacteria. Specifically, the compounds of the presentinvention have significant improvement in their microbiological spectrumof activity by having improved inhibition of Gram negative and/or Grampositive bacteria such as H. influenza and S pneumonia. For examplewhere, in one example, the average comparative index (ACI) is greaterthan 3 dilution steps for the improved inhibition of H. influenza andadditionally shows an ACI of 0.4 dilution steps for the improvedinhibition of S. pneumonia. In another example, the ACI is 3 dilutionsteps for the improved inhibition of S. pneumonia and additionally showsan ACI of 1.2 dilution steps for the improved inhibition of H.influenza.

The compounds of the invention also preferably have improved safety,toxicity and pharmacokinetic properties, e.g. a decrease or eliminationof potential adverse events in human relative to prior art compounds.

In one aspect, compositions, for treating or preventing infectiousdisorders are provided, comprising a compound of the invention, apharmaceutically acceptable salt thereof or a prodrug thereof, asdisclosed herein in combination with a pharmaceutically acceptablecarrier. In another embodiment, such compositions further includeanother therapeutic agent.

In another aspect, there is provided a dosage amount of a compound ofthe invention, a pharmaceutically acceptable salt thereof or a prodrugthereof, as disclosed herein in an effective amount for the treatment,prevention or alleviation of a disorder, such as an infectious disorder.These compounds or derivatives thereof can be screened for activityagainst different microbial agents and appropriate dosages can bedetermined using methods available in the art.

The compounds of the invention can be used to treat a subject to treat,prevent, or reduce the severity of an infection. Subjects includeanimals, plants, blood products, cultures and surfaces such as those ofmedical or research equipment, such as glass, needles, surgicalequipment and tubing, and objects intended for temporary or permanentimplantation into an organism. Preferred animals include mammals, e.g.,mice, rats, cats, dogs, cows, sheep, pigs, horses, swine, primates, suchas rhesus monkeys, chimpanzees, gorillas, and most preferably humans.Treating a subject includes, but is not limited to, preventing,reducing, or eliminating the clinical symptoms caused by an infection ofa subject by a microorganism; preventing, reducing, or eliminating aninfection of a subject by a microorganism; or preventing, reducing, oreliminating contamination of a subject by a microorganism. Themicroorganism involved is preferably a prokaryote, more preferably abacterium.

In one aspect, methods of treating or preventing an infectious disorderin a subject, such as a human or other animal subject, that areresponsive to inhibition of peptidyl deformylase are provided, byadministering to the subject an effective peptidyl deformylaseinhibiting amount of a compound of the invention, a pharmaceuticallyacceptable salt thereof or a prodrug thereof. In one embodiment, thecompound or its derivative is administered in a pharmaceuticallyacceptable form optionally in a pharmaceutically acceptable carrier. Thecompound of the invention, pharmaceutically acceptable salt thereof orprodrug thereof, can be administered alone or in combination withanother therapeutic agent. Examples of such therapeutic agents include,but are not limited to, β-lactam, quinolone, macrolide, glycopeptide andoxazolidinone. As used herein, an “infectious disorder” is any disordercharacterized by the presence of a microbial infection, such as thepresence of bacteria. Such infectious disorders include, for example,central nervous system infections, external ear infections, infectionsof the middle ear, such as acute otitis media, infections of the cranialsinuses, eye infections, infections of the oral cavity, such asinfections of the teeth, gums and mucosa, upper respiratory tractinfections, lower respiratory tract infections, genitourinaryinfections, gastrointestinal infections, gynecological infections,septicemia, bone and joint infections, skin and skin structureinfections, bacterial endocarditis, burns, antibacterial prophylaxis ofsurgery, antibacterial prophylaxis in immunosuppressed patients, such aspatients receiving cancer chemotherapy, or organ transplant patients andchronic diseases caused by infectious organisms, e.g., arteriosclerosis.The compounds and compositions comprising the compounds can beadministered by routes such as topically, locally or systemically.Systemic application includes any method of introducing the compoundinto the tissues of the body, e.g., intrathecal, epidural,intramuscular, transdermal, intravenous, intraperitoneal, subcutaneous,sublingual, nasal, vaginal, rectal, and oral administration. Thespecific dosage of antimicrobial to be administered, as well as theduration of treatment, can be adjusted as needed.

In another aspect of the present invention, methods are provided forinhibiting peptidyl deformylase. In one embodiment, the method comprisesadministering to a subject in need thereof an effective peptidyldeformylase inhibiting amount of a compound of formula (I), apharmaceutically acceptable salt thereof or a prodrug thereof. The terms“subject” and “effective peptidyl deformylase inhibiting amount” are asdefined above.

In yet another aspect of the invention, there is also provided the useof a compound of the formula (I) as defined above, a pharmaceuticallyacceptable salt thereof or a prodrug thereof in the preparation of amedicament for use in the treatment of diseases mediated by peptidyldeformylase.

Administration and Pharmaceutical Composition

The present invention also provides pharmaceutical compositions whichcomprise a bioactive N-formyl hydroxylamine compound, a pharmaceuticallyacceptable salt thereof, or a prodrug thereof and a pharmaceuticallyacceptable carrier. The compositions of the invention include those in aform adapted for oral, topical or parenteral use and can be used for thetreatment of bacterial infection in a subject such as animals,preferably, mammals, more preferably, humans. The pharmaceuticalcompositions can further include another therapeutic agent as describedbelow.

The antibiotic compounds, also referred to herein as antimicrobialcompounds, according to the invention can be formulated foradministration in any convenient way for use in human or veterinarymedicine, by analogy with other antibiotics. Such methods are known inthe art (see, e.g., Remington's Pharmaceutical Sciences, Easton, Pa.:Mack Publishing Co.) and are not described in detail herein.

The composition can be formulated for administration by any route knownin the art, such as subdermal, inhalation, oral, topical or parenteral.The compositions can be in any form known in the art, including but notlimited to tablets, capsules, wafers, fast melts (without wafers),powders, granules, lozenges, creams or liquid preparations, such as oralor sterile parenteral solutions or suspensions. The compounds can alsobe administered in liposomal, micellar or microemulsion formulations.The compounds can also be administered as prodrugs, where the prodrugadministered undergoes biotransformation in the treated mammal to a formwhich is biologically active.

The topical formulations of the present invention can be presented as,for instance, ointments, creams or lotions, solutions, salves,emulsions, plasters, eye ointments and eye or ear drops, impregnateddressings, transdermal patches, sprays and aerosols, and can containappropriate conventional additives such as preservatives, solvents toassist drug penetration and emollients in ointments and creams.

The formulations can also contain compatible conventional carriers, suchas cream or ointment bases and ethanol or oleyl alcohol for lotions.Such carriers can be present, for example, from about 1% up to about 99%of the formulation. For example, they can form up to about 80% of theformulation.

Tablets and capsules for oral administration can be in unit dosepresentation form, and can contain conventional excipients such asbinding agents, for example, syrup, acacia, gelatin, sorbitol,tragacanth, or polyvinylpyrollidone; fillers, for example, lactose,sugar, maize-starch, calcium phosphate, sorbitol or glycine; tablettinglubricants, for example, magnesium stearate, talc, polyethylene glycolor silica; disintegrants, for example, potato starch; or acceptablewetting agents, such as sodium lauryl sulphate. The tablets can becoated according to methods well-known in standard pharmaceuticalpractice.

Oral liquid preparations can be in the form of, for example, aqueous oroily suspensions, solutions, emulsions, syrups or elixirs, or can bepresented as a dry product for reconstitution with water or anothersuitable vehicle before use. Such liquid preparations can containconventional additives, such as suspending agents, for example,sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminium stearate gel orhydrogenated edible fats; emulsifying agents, for example, lecithin,sorbitan monooleate, or acacia; non-aqueous vehicles (which can includeedible oils), for example, almond oil, oily esters such as glycerine,propylene glycol, or ethyl alcohol; preservatives, for example, methylor propyl p-hydroxybenzoate or sorbic acid, and, if desired,conventional flavoring or coloring agents.

For parenteral administration, fluid unit dosage forms are preparedutilizing the compound and a sterile vehicle, water being preferred. Thecompound, depending on the vehicle and concentration used, can be eithersuspended or dissolved in the vehicle or other suitable solvent. Inpreparing solutions, the compound can be dissolved in water forinjection and filter sterilized before filling into a suitable vial orampule and sealing. Advantageously, agents such as a local anestheticpreservative and buffering agents can be dissolved in the vehicle. Toenhance the stability, the composition can be frozen after filling intothe vial and the water removed under vacuum. The dry lyophilized powderis then sealed in the vial and an accompanying vial of water forinjection can be supplied to reconstitute the liquid prior to use.Parenteral suspensions are prepared in substantially the same mannerexcept that the compound is suspended in the vehicle instead of beingdissolved and sterilization cannot be accomplished by filtration. Thecompound can be sterilized by exposure to ethylene oxide beforesuspending in the sterile vehicle. Advantageously, a surfactant orwetting agent is included in the composition to facilitate uniformdistribution of the compound.

The compositions can contain, for example, from about 0.1% by weight toabout 99% by weight, e.g., from about 10-60% by weight, of the activematerial, depending on the method of administration. Where thecompositions comprise dosage units, each unit will contain, for example,from about 1-1000 mg of the active ingredient. The dosage as employedfor adult human treatment will range, for example, from about 1-3000 mgper day, for instance 1500 mg per day depending on the route andfrequency of administration. Such a dosage corresponds to about 0.015-50mg/kg per day. Suitably the dosage is, for example, from about 5-20mg/kg per day.

Representative pharmaceutical formulations containing a compound offormula (I) are described below.

The present invention also provides a process for preparing a compoundof the invention, e.g. a compound of formula (I) which process comprisesreacting a compound of formula (V)

wherein R₁ and R₂ are as defined above and Y is a hydroxy protectinggroup, or a functional derivative thereof, with a compound of formula(VI)

wherein R₃ and R₄ are as defined above, and n is equal to 1, and whererequired, converting the resulting compounds obtained in free form intosalt forms or vice versa.

Functional derivatives of compounds of formula (V) include e.g. acidchloride, acid anhydride or an activated ester.

Above reactions may be carried out according to methods known in the artor as disclosed in the Examples below. The reaction may conveniently becarried out in the presence of a base and then followed byhydrogenation, preferably in the presence of a hydrogenation catalyst.Suitable bases include e.g. Hunig base (i.e. diisopropylethylamine) andinorganic bases such as sodium bicarbonate. The hydrogenation catalyst,preferably a palladium catalyst, e.g. palladium on carbon or palladiumblack, may then be added to the resulting product, e.g. afterconcentration and stirred under a hydrogen atmosphere e.g. for about 16to about 24 hours. The palladium catalyst may be added preferably fromabout 5 mol % to about 10 mol % of the concentrated product.

Compounds of formula (V), used as starting materials, may be preparede.g. by reacting a compound of formula (VII)

wherein R₁, R₂, and Y are as defined above, e.g. under mild basicconditions e.g. as known in the art. Typically, this reaction may becarried out by dissolving the compound of formula (VII) e.g. in amixture of an inert solvent, such as THF, DMF, toluene, dioxane orCH₂Cl₂, and water, and adding hydrogen peroxide and then an aqueoussolution of the base in water to the cooled mixture. Examples of baseinclude, e.g. sodium bicarbonate, lithium hydroxide, sodium hydroxideand the like. The base may be used preferably at from about 1.1 to about1.5 equivalents to the compound of formula (VII).

Compounds of formula (VII) may be produced e.g. by reacting a compoundof formula (VIII) wherein R₁, R₂, and Y are as defined above, withformic acid as known in the art. The reaction may typically be carriedout, e.g. at 0° C., by adding a solution of acetic anhydride in formicacid to a solution of a compound of formula (VIII) in formic acid.

Compounds of formula (VIII) may be prepared e.g. by reacting a compoundof formula (IX) wherein R₁, R₂, and Y are as defined above, with asolution of p-toluenesulfonic acid in an inert organic solvent, and asolution of Na₂CO₃, e.g. 1M, as known in the art.

Compounds of formula (IX) may be prepared e.g. by reacting a compound offormula (X) wherein R₁ is as defined above, with a hydroxy protectedcompound of formula (XI) wherein Y is aryl, alkyl, aralkyl or silyl, asknown in the art.

The compound of formula (X) may be produced e.g. by reacting a compoundof formula (XII) with pivaloyl chloride, wherein R₄ is as defined above,as known in the art.

Insofar as the production of starting materials is not particularlydescribed, the compounds are known or may be prepared analogously tomethods known in the art or as disclosed in the examples hereinafter.

All patents, patent applications and publications cited in thisapplication are hereby incorporated by reference in their entirety forall purposes to the same extent as if each individual patent, patentapplication or publication were so individually denoted.

The following abbreviations are used:

AcOH, HOAc=acetic acidAc₂O=acetic anhydrideBOC, Boc=t-butyloxycarbonylDCM=dichloromethaneDIEA=diisopropylethylamineDMF=dimethylformamideDMSO=dimethylsulfoxideEt=ethylEtOAc=ethyl acetateFmoc, FMOC=9-fluorenylmethyloxycarbonylHATU=O-(7-aza-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphateMCPBA=meta-chloroperoxy-benzoic acidMe=methylMeOH=methanolMMP=matrix metalloproteinaseNVOM=nitroveratryloxymethyl etherp-TSA=p-toluenesulfonic acidRT=room temperatureTFA=trifluoroacetic acidtBu=t-butylTHF=tetrahydrofuranTHP=2-tetrahydropyranylTsOH or p-TSA=toluenesulfonic acid

General Synthetic Scheme

Compounds of this invention can be made by the methods depicted in thereaction schemes shown below.

The starting materials and reagents used in preparing these compoundsare either available from commercial suppliers such as Aldrich ChemicalCo., (Milwaukee, Wis., USA), Bachem (Torrance, Calif., USA),Emka-Chemie, or Sigma (St. Louis, Mo., USA) or are prepared by methodsknown to those skilled in the art following procedures set forth inreferences such as Fieser and Fieser's Reagents for Organic Synthesis,Volumes 1-15 (John Wiley and Sons, 1991); Rodd's Chemistry of CarbonCompounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers,1989), Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991),March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition),and Larock's Comprehensive Organic Transformations (VCH Publishers Inc.,1989). These schemes are merely illustrative of some methods by whichthe compounds of this invention can be synthesized, and variousmodifications to these schemes can be made and will be suggested to oneskilled in the art having referred to this disclosure.

The starting materials and the intermediates of the reaction may beisolated and purified if desired using conventional techniques,including but not limited to filtration, distillation, crystallization,chromatography, and the like. Such materials may be characterized usingconventional means, including physical constants and spectral data.

Preparation of Compounds of Formula (I)

Compounds of formula (I) can be prepared by methods well known in theart of organic chemistry. Representative synthetic procedures forpreparing compounds of the present invention are illustrated anddescribed in detail below. For example, compounds of formula (I) can beprepared as described in Schemes A-B below.

General Procedure A Synthesis of1-{2(R)-[(formylhydroxyamino)-methyl]-alkanoyl}-pyrrolidine-2(S)-carboxylicacid amide

Step 1: 2-n-butyl acrylic acid (A-2)

To a solution of alkyl malonic acid A-1 (R=n-butyl (107.4 mmol) inethanol (200 mL) is added piperidine (12.7 mL, 128.8 mmol, 1.2 equiv.)and 37% aqueous formaldehyde (40.0 mL, 536.9 mmol, 5 equiv.). Thesolution is heated to 80° C. during which time a precipitate appears,and then gradually redissolves over 1 hour. The reaction mixture isstirred at 80° C. overnight then cooled to room temperature (rt). Thesolvents are removed under reduced pressure, and the residue isdissolved in ethyl acetate, washed successively with 1 M HCl and brine,dried over anhyd Na₂SO₄, and filtered. The filtrate is concentrated togive the title compound A-2 as a clear oil.

Step 2: 4-benzyl-3-(2-butyl-acryloyl)-oxazolidin-2-one (A-3)

2-n-Butyl acrylic acid (9.90 g, 77.2 mmol, and 1 equiv.) is dissolved indry THF (260 mL) and cooled to −78° C. under a blanket of nitrogen.Hunig's base (17.5 mL, 100.4 mmol, 1.3 equiv.) and pivaloyl chloride(9.5 mL, 77.2 mmol, 1 equiv.) are added at such a rate that thetemperature remained below −60° C. The mixture is stirred at −78° C. for30 minutes, warmed to rt for 2 hours, and finally cooled back to −78° C.

In a separate flask, (S)-(−)-4-benzyl-2-oxazolidinone (13.49 g, 77.24mmol) is dissolved in dry THF (150 mL) and cooled to −78° C. under ablanket of nitrogen. n-Butyllithium (2.5 M solution in hexanes, 30.9 mL,77.2 mmol, 1 equiv.) is added slowly at −78° C., and the mixture isstirred for 30 minutes at rt. The resulting anion is slowly transferredvia a cannula into the original reaction vessel. The mixture is allowedto warm to rt and is stirred overnight at rt. The reaction is quenchedwith 1 M KHCO₃, and the solvents are removed under reduced pressure. Theresidue is partitioned between ethyl acetate and water. The organiclayer is washed with brine, dried over anhydrous Na₂SO₄, filtered, andconcentrated to give a yellow oil which is purified by flashchromatography (hexane:ethyl acetate=4:1) to yield the title compoundA-3 as a white solid (15.0 g, 52.2 mmol, 68%).

Step 3: 4-benzyl-3-[2-(benzyloxyamino-methyl)-hexanoyl]-oxazolidin-2-one(p-toluenesulfonic acid salt)

Compound A-3 (8.25 g, 28.7 mmol) is mixed with O-benzylhydroxylamine(7.07 g, 57.4 mmol, 2 equiv.) and stirred for 40 hours at rt undernitrogen. The mixture is dissolved in ethyl acetate andp-toluenesulfonic acid (21.84 g, 114.8 mmol, and 4 equiv.) is added toprecipitate excess O-benzylhydroxylamine as a white solid. The whitesolid is filtered off, and the filtrate is concentrated to give a crudeyellow oil (HPLC analysis indicated a small trace of starting material).Charging the crude yellow oil with excess diethyl ether and cooling to0° C. for 30 minutes gives a solid which is collected by filtration anddried in vacuo to afford the title compound as a white crystalline solid(single diastereomer).

Step 4: 4-benzyl-3-[2-(benzyloxyamino-methyl)-hexanoyl]-oxazolidin-2-one(A-5)

To a solution of p-TSA salt (22.9 g, 39 3 mmol) dissolved in ethylacetate (400 mL), was added 1 M Na₂CO₃ (200 mL, 5 equiv.) and stirred atrt for 30 minutes. The layers were separated, and the aqueous layerextracted with ethyl acetate. The combined organic layers were driedover anhyd Na₂SO₄, filtered, and concentrated to give the title compoundas a pale opaque oil (15.8 g, 38.6 mmol, 98%).

Step 5:N-[2-(4-benzyl-2-oxo-oxazolidine-3-carbonyl)-hexyl]-N-benzyloxy-formamide(A-6)

A solution of compound A-5 (5.38 g, 13.1 mmol, 1 equiv.) in formic acid(7.4 mL, 196.6 mmol, 15 equiv.) is cooled to 0° C. under a blanket ofnitrogen. In a separate flask, formic acid (7.4 mL, 196.6 mmol, 15equiv.) is cooled to 0° C. under a blanket of nitrogen, and aceticanhydride (2.47 mL, 26.2 mmol, 2 equiv.) is added dropwise. The solutionis stirred at 0° C. for 15 minutes. The resulting mixed anhydride isslowly transferred via syringe into the original reaction vessel. Themixture is stirred at 0° C. for 1 hour, then at rt for 3 hours. Themixture is concentrated, taken up in dichloromethane, and washedsuccessively with saturated NaHCO₃ and brine. The organic layer is driedover anhydrous Na₂SO₄, filtered, and concentrated to give an opaque oilwhich is purified by flash chromatography (hexane:ethyl acetate=2:1 thendichloromethane:acetone=9:1) to yield the title compound as a colorlessoil.

Step 6: 2-[(benzyloxy-formyl-amino)-methyl]-hexanoic acid (A-7)

Compound A-6 (0.163 g, 0.372 mmol, 1 equiv.) is dissolved in THF (4.5mL) and water (1.5 mL) and cooled to 0° C. Hydrogen peroxide (30% inwater, 228 μL, 2.23 mmol, 6 equiv.) is added dropwise followed by theslow addition of a solution of lithium hydroxide (0.019 g, 0.446 mmol,1.2 equiv.) in water (350 μL). The resulting mixture is stirred at 0° C.for 1.5 hours. The basic reaction mixture is quenched with AmberliteIR-120 resin (H⁺) to pH 4-5 at 0° C. The resin is filtered off andrinsed with ethyl acetate. The mixture is concentrated to remove THF,and then taken up in ethyl acetate. The aqueous layer is separated, andthe organic layer dried over anhydrous Na₂SO₄, filtered, andconcentrated to give an opaque oil which was purified by flashchromatography (dichloromethane:acetone=4:1 then acetone: methanol=99:1)to yield the title compound A-7 as a colorless oil.

Step 7:1-{2-[(benzyloxy-formyl-amino)-methyl]-hexanoyl}-pyrrolidine-2-carboxylicacid amide

To a solution of compound A-7 (0.190 g, 0.680 mmol, 1 equiv.) in drydioxane (4 mL) at rt under nitrogen is added successively Hunig's base(391 μL, 2.24 mmol, 3.3 equiv.), amine A-8 (0.748 mmol, 1.1 equiv.) andHATU (0.284 g, 0.748 mmol, 1.1 equiv.). The resulting mixture is stirredat rt for 22 hours. The mixture is partitioned between ethyl acetate and10% citric acid. The organic layer is washed with brine and saturatedNaHCO₃, dried over anhydrous Na₂SO₄, filtered, and concentrated. Theresidue is purified by flash chromatography(dichloromethane:acetone=3:1) to give the title compound as a colorlessoil.

Step 2:1{2-[(formyl-hydroxy-amino)-methyl]-hexanoyl}-pyrrolidine-2-carboxylicacid amide (A-9)

Pd—C (0.059 g, 0.1 equiv.) is added to a solution of above compound(0.550 mmol, 1 equiv.) in a 1:1 ethyl acetate/ethanol solution (12 mL)under a blanket of nitrogen. The mixture is stirred under hydrogenatmosphere for 36 hours. The catalyst is removed by filtration through apad of Celite. The filtrate is concentrated, and the residue waspurified by preparative TLC (dichloromethane:acetone=2:1) to give thetitle compound as an amorphous solid (0.121 g, 0.334 mmol, 61%).

General Procedure B Synthesis of1-{2(R)-[(formylhydroxyamino)-methyl]-alkanoyl}-pyrrolidine-2(S)-carboxylateester

Step 1:1-{2-[(benzyloxy-formyl-amino)-methyl]-hexanoyl}-pyrrolidine-2-carboxylicacid ester

To a solution of compound A-7 (0.680 mmol, 1 equiv.) in dry dioxane (4mL) at rt under nitrogen is added successively Hunig's base (391 μL,2.24 mmol, 3.3 equiv.), amine A-10 (0.748 mmol, 1.1 equiv.) and HATU(0.284 g, 0.748 mmol, 1.1 equiv.). Usual work-up and purificationprovides the title compound.

Step 2:1{2-[(formyl-hydroxy-amino)-methyl]-hexanoyl}-pyrrolidine-2-carboxylicacid ester (A-11)

Pd—C (0.059 g, 0.1 equiv.) is added to a solution of above compound(0.550 mmol) in a 1:1 ethyl acetate/ethanol solution (12 mL) under ablanket of nitrogen. The mixture is stirred under hydrogen atmospherefor 36 hours. The catalyst is removed by filtration through a pad ofCelite. The filtrate is concentrated, and the residue is purified bypreparative TLC (dichloromethane:acetone=2:1) to give the titlecompound.

General Procedure C Preparation of pyrrolidine-2-S-carboxylic acidpyridin-2-ylamide (A-8) (X=CH₂, n=1, R₁=2-pyridyl) Step 1:2-S-(pyridin-2-ylcarbamoyl)-pyrrolidine-1-carboxylic acid benzyl ester

A solution of Z-prochloride (5.0 g, 18.7 mmol, 1 equiv.) in pyridine (40mL) is cooled to 0° C. under a blanket of nitrogen. 2-aminopyridine(5.27 g, 56.0 mmol, 3 equiv.) in pyridine (10 mL) is added dropwise. Theresulting mixture is stirred at rt for 4 hours, then concentrated. Theresidual oil is dissolved in ethyl acetate and washed successively withwater, 10% citric acid, saturated NaHCO₃, and brine. The organic layeris dried over anhydrous Na₂SO₄, filtered, and concentrated to give thetitle compound (4.21 g, 13.0 mmol, 69%) as an opaque solid.

Step 2: Pyrrolidine-2-S-carboxylic acid (pyridin-2-yl) amide hydrobromicacid salt

A solution of above compound (4.21 g, 13.0 mmol, 1 equiv.) in aceticacid (65 mL) at rt is treated with HBr (5.7 M, 33% in acetic acid, 110mL, 649 mmol, 50 equiv.), and the mixture is stirred at rt for 2 hours.Charging the reaction mixture with excess diethyl ether and cooling to0° C. for 30 minutes gives a solid which is collected by filtration anddried in vacuo to afford the title compound as a brownish powder.

General Procedure D 4-R-hydroxy-pyrrolidine-2-S-carboxylic acid(5-methyl-pyridine-2-yl)-amide

The coupling of O-tert butyl protected proline (1 mmol) with 5-picoline(1.5 mmol) in DMF (5 mL) under HATU (1.3 mmol) and N,N-diisopropylethylamine (5 mmol) condition followed by removal of O-tert butyl withTFA-dichloroethane (1:1) provides the title compound in 85% yield.

General Procedure E 4-S-fluoro-pyrrolidine-2-S-carboxylic acid(5-methyl-pyridine-2-yl)-amide

The above hydroxy compound (2 mmol) in methylenechloride (20 mL) istreated with N′N-diethylamino sulphur trifluoride (DAST; 4 mmol) at −70°C. Then, reaction mixture is allowed to stir at rt for 16 hours andwashed with cold aq. sodium bicarbonate solution, dried and concentratedunder reduced pressure. It is purified on silica gel columnchromatography to give N-protected derivative which on treatment withHBr—AcOH provides an amino compound

General Procedure F 4-S-hydroxy-pyrrolidine-1,2-dicarboxylic acid1-tert-butyl ester-2-methyl ester

To a solution of trans-4-hydroxy compound (1 mmol), triphenyl phosphine(1.5 mmol) and benzoic acid (1.5 mmol) in THF (10 mL) is addedN,N-diisopropyl-azo dicarboxylate (1.5 mmol) in THF (5 mL) dropwise at0° C. It is allowed to stir at rt for 16 hours. The solvent is removedunder reduced pressure and residue is dissolved in ether. It is icecooled to precipitate phosphine oxide which is removed by filtration andfiltrate is concentrated under reduced pressure. The crude material istreated with methanolic sodium methoxide for 2 hours at 0° C. to givetitle cis-hydroxy compound.

General Procedure G 4-R-fluoro-pyrrolidine-2-S-carboxylic acid(5-methyl-pyridine-2-yl)-amide

The fluorination of the above cis-hydroxy provides the trans-4-fluoroderivative which on saponification gives the corresponding acid. Theamine is prepared from 4-R-fluoro-pyrrolidine-1-carboxylic acidtert-butyl ester and 5-methyl-pyridin-2-ylamine under HATU condition togive proline amide derivative which on treatment with 4 M HCl in dioxaneprovides the desired amine.

EXAMPLE 11-[2-Cyclopentylmethyl-3-(formyl-hydroxy-amino)-propionyl]-pyrrolidine-2-carboxylicacid (5-fluoro-1-oxy-pyridin-2-yl)-amide

The title compound is prepared according to General Procedure A from3-benzyloxy-formyl-amino)-2-cyclopentylmethyl-propionic acid A-7(R=cyclopentylmethyl) and pyrrolidine-2-carboxylic acidpyridin-2-ylamide A-8 (X=CH₂, n=1, R₁=5-Fluoro 2-pyridyl).

2-Cyclopentylmethyl-3-[formyl-(tetrahydro-pyran-2-yloxy)-amino]-propionicacid

Is prepared from cyclopentylmethyl malonic acid as described below.

Bromomethyl-cyclopentane

A solution of cyclopentane methanol (48.5 g, 484 mmol), Et₃N (88.0 mL,631 mmol), and anhydrous THF (1 L) is cooled to 4° C., and stirred undernitrogen. Methanesulfonyl chloride (45.0 mL, 581 mmol) is slowly addedto the stirring solution, while maintaining 10° C. The mixture isstirred for an additional hour at 10° C., and LiBr (300.0 g, 3454 mmol)is slowly added (exothermic). The reaction mixture is stirred for anadditional 16 hours at room temperature. Water is added to dissolve thesalt, and the mixture is extracted with Et₂O. The Et₂O layers arecombined, dried over Na₂SO₄, and is carefully concentrated (25° C. at100 torr). The crude product is purified by vacuum distillation (35° C.at 1 torr, the desired compound is the first fraction to be collected).This gives bromomethyl-cyclopentane (31.4 g, 40% yield) as a colorlessoil.

2-Cyclopentylmethyl-malonic acid

A solution of diethyl malonate (36.91 g, 230.4 mmol), anhydrous methanol(400 mL), and NaOMe (25% in methanol, 49.79 g, 230.4 mmol) is stirred atreflux for one hour under nitrogen. Bromomethyl-cyclopentane (31.31 g,192.0 mmol) is added to the mixture, and stirred for an additional 3hours. A solution of NaOH (23.04 g, 576.0 mmol) in water (400 mL) isadded, and the mixture is stirred for an additional 1 hour at reflux.The mixture is cooled, diluted with water, and extracted with ether. Theether layer is discarded, and the aqueous layer is acidified with 1N HClto pH=1. The aqueous layer is extracted with EtOAc. The EtOAc layers arecombined, dried over Na₂SO₄, and concentrated. This gives2-cyclopentylmethyl-malonic acid (21.0 g, 59% yield) as a white solid.

2-Cyclopentylmethyl-acrylic acid

A mixture of 2-cyclopentylmethyl-malonic acid (24.90 g, 133.7 mmol),piperidine (15.9 mL, 160.8 mmol), 37% aqueous formaldehyde (51.0 mL,647.2 mmol), and EtOH (250 mL) is stirred at reflux for 16 hours. Thereaction is quenched with 1N HCl to a pH=1, and the mixture is extractedwith EtOAc. The EtOAc layers are combined, dried over Na₂SO₄, andconcentrated. The crude product is purified by flash chromatography(SiO₂, 10% acetone in DCM), which gives 2-cyclopentylmethyl-acrylic acid(17.65 g, 86% yield) as an oil.

4-Benzyl-3-(2-cyclopentylmethyl-acryloyl)-oxazolidin-2-one

2-Cyclopentylmethyl-acrylic acid (17.65 g, 114.5 mmol) is dissolved inanhydrous THF (200 mL) and cooled to −78° C. under nitrogen.N,N-Diisopropylethylamine (25.9 mL, 148.7 mmol) and trimethylacetylchloride (14.1 mL, 114.5 mmol) are added consecutively at such a ratethat the temperature remained below −60° C. and that gas evolution iscontrolled. The mixture is stirred at −78° C. for 30 minutes, stirred atroom temperature for 2 hours, and cooled back down to −78° C.

In a separate flask, (S)-(−)-4-benzyl-2-oxazolidinone (20.30 g, 114.6mmole) is dissolved in anhydrous THF (400 mL) and cooled to −78° C.under nitrogen. BuLi (2.5M, 45.8 mL, 114.5 mmole) is slowly added at−78° C., and the mixture is stirred for 30 minutes at room temperature.The resulting anion is slowly transferred via a cannula into theoriginal reaction vessel. The mixture is allowed to warm to roomtemperature, and is stirred overnight at room temperature (16 hours).The reaction mixture is quenched with 1M KHCO₃, and is extracted withEtOAc. The organic layers are combined, washed with brine, dried overNa₂SO₄, and concentrated to give a yellow oil. The crude product ispurified by flash chromatography (SiO₂, 20% EtOAc in hexane) to give4-benzyl-3-(2-cyclopentylmethyl-acryloyl)-oxazolidin-2-one (22.9 g, 64%)of as an oil.

4-Benzyl-3-[2-cyclopentylmethyl-3-(tetrahydro-pyran-2-yloxyamino)-propionyl]-oxazolidin-2-one

4-Benzyl-3-(2-cyclopentylmethyl-acryloyl)-oxazolidin-2-one (22.90 g,73.1 mmol) and O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (34.24 g,292.3 mmol) is combined at stirred at 45° C. for 48 hours undernitrogen. The crude product is purified by flash chromatography (SiO₂,0→30% EtOAc in hexane), which gives4-benzyl-3-[2-cyclopentylmethyl-3-(tetrahydro-pyran-2-yloxyamino)-propionyl]-oxazolidin-2-one(21.65 g, 69% yield) as an oil.

N-[3-(4-Benzyl-2-oxo-oxazolidin-3-yl)-2-cyclopentylmethyl-3-oxo-propyl]-N-(tetrahydropyran-2-yloxy)-formamide

A mixture of formic acid (45.0 mL, 1193 mmol) and acetic anhydride (90.0mL, 952 mmol) is stirred at 50° C. for one hour under nitrogen. A secondflask is charged with4-benzyl-3-[2-cyclopentylmethyl-3-(tetrahydro-pyran-2-yloxyamino)-propionyl]-oxazolidin-2-one(21.62 g, 50.2 mmol), Et₃N (170.0 mL, 1220 mmol), and anhydrous DCM (450mL). This second mixture is cooled to 4° C. under nitrogen, and themixed acid solution is slowly added to the second flask, whilemaintaining 10° C. The combined mixture is stirred for 30 minutes at 10°C., quenched with saturated, washed with aqueous NaHCO₃ solution, andextracted with DCM. The DCM layers are combined, dried over Na₂SO₄, andconcentrated. The crude product is purified by flash chromatography(SiO₂, 50% EtOAc in hexane), which givesN-[3-(4-benzyl-2-oxo-oxazolidin-3-yl)-2-cyclopentylmethyl-3-oxo-propyl]-N-(tetrahydro-pyran-2-yloxy)-formamide(20.10 g, 87% yield) as an oil.

2-Cyclopentylmethyl-3-[formyl-(tetrahydro-pyran-2-yloxy)-amino]-propionicacid

N-[3-(4-Benzyl-2-oxo-oxazolidin-3-yl)-2-cyclopentylmethyl-3-oxo-propyl]-N-(tetrahydro-pyran-2-yloxy)-formamide(3.65 g, 7.96 mmol), THF (125 mL), and water (40 mL) is cooled to 4° C.To this mixture, is added 30% H₂O₂ (5.2 mL, 50.90 mmole) and LiOHmonohydrate (0.40 g, 9.53 mmol), respectively. The reaction mixture isstirred for 1.5 hours. The mixture is slowly quenched with 0.5 M Na₂SO₃,while maintaining the temperature below 15° C. with an ice bath. Thequenched mixture is stirred for an addition 30 minutes, concentrated invacuo until the THF solvent is removed, and washed with EtOAc. The basicreaction mixture is acidified with Amberlite IR-120 resin (H+) topH=4.5. Brine is added to the acidic solution, and the combined mixtureis extracted with EtOAc. The organic layers from the acidic solutionwashing are combined, dried over Na₂SO₄, and concentrated in vacuo. Thisgave2-cyclopentylmethyl-3-[formyl-(tetrahydro-pyran-2-yloxy)-amino]-propionicacid (1.20 g, 50% yield) as an oil.

3-(Benzyloxy-formyl-amino)-2-cyclopentylmethyl-propionic acid

Is prepared from 2-Cyclopentylmethyl-acrylic acid and O-benzylhydroxamine as described for the synthesis of the corresponding O-THpprotected building block.

4-Benzyl-3-(3-benzyloxyamino-2-cyclopentylmethyl-propionyl)-oxazolidin-2-one

N-[3-(4-Benzyl-2-oxo-oxazolidin-3-yl)-2-cyclopentylmethyl-3-oxo-propyl]-N-benzyloxy-formamide(compound A-G, where: R₁=cyclopenylmethyl, PG₁=benzyl)

3-(Benzyloxy-formyl-amino)-2-cyclopentylmethyl-propionic acid

1-[3-Benzyloxy-formyl-amino)-2-cyclopentylmethyl-propionyl]-pyrrolidine-2-carboxylicacid (5-fluoro-1-oxy-pyridin-2-yl)-amide

EXAMPLE 24-Fluoro-1-{2-[(formyl-hydroxy-amino)-methyl]-hexanoyl}-pyrrolidine-2-carboxylicacid (5-fluoro-1-oxy-pyridin-2-yl)-amide

The title compound is prepared according to General Procedure A from2-[(benzyloxy-formyl-amino)-methyl]-hexanoic acid A-7 (R=n-butyl) andpyrrolidine-2-carboxylic acid pyridin-2-ylamide A-8 (X=CHF n=1,R₁=5-Fluoro 2-pyridyl).

4-trans-fluoro-pyrrolidine-2-carboxylicacid-[2-amino-5-fluoro-pyridin-2-yl]amide

To a DMF solution (15 mL) of Boc-L-Pro-4-F—OH (2.5 g, 10.73 mmol, 1equiv) Hunig's base (Diisopropylethylamine, abbrev. DIEA) (6.73 mL,38.61 mmol, 3.6 eq) is added and the mixture cooled to 0° C. This isfollowed by the addition of 2-Amino-5-fluoro pyridine (1.44 g, 12.87mmol, 1.2 equiv), and HATU (4.89 g, 12.87 mmol, 1.2 equiv) at 0° C. Theresulting mixture is stirred at room temperature for 16 h. The mixtureis partitioned between excess ethyl acetate and 10% citric acid. Theorganic layer is washed with brine and sat. NaHCO₃, dried over anhydrousNa₂SO₄, filtered, and concentrated. The residue is purified by silicagel chromatography (Hexanes:Ethyl acetate=1:0-7:3) to give the titlecompound as a colorless syrup (2.5 g, 71%).

4-trans-fluoro-pyrrolidine-2-carboxylicacid-[2-amino-5-fluoro-pyridin-2-yl]amide (hydrochloric acid salt)

The Boc-proline-4-fluoro-pyridine amide (1 g, 3.06 mmol, 1 equiv) istreated with 4N HCl/dioxane (30 mL, 120 mmol, 40 equiv) at roomtemperature and allowed to stir for 16 h. The mixture is concentrated,and the residue was coevaporated with toluene 2×, and concentrated togive a purplish pink solid (1 g).

1-{2-[(Benzyloxy-formyl-amino)-methyl]-hexanoyl}-4-trans-fluoro-pyrrolidine-2-carboxylicacid-(2-amino-5-fluoro-pyridin-2-yl)-amide

To a DMF solution (10 mL) of trans-fluoro-proline-5-fluoro-aminopyridineamide HCl salt (644 mg, 2.15 mmol, 1.2 equiv), are successively addedHunig's base (2 mL, 10.8 mmol, 5 equiv), Versiacid VRI 172 (500 mg, 1.79mmol, 1 equiv), and HATU (818 mg, 2.15 mmol, 1.2 equiv) at 0° C. Theresulting mixture is stirred at room temperature for 16 h. The mixtureis partitioned between excess ethyl acetate and 10% citric acid. Theorganic layer is washed with brine and sat. NaHCO₃, dried over anhydrousNa₂SO₄, filtered, and concentrate. The compound is purified by silicagel chromatography in DCM:Acetone (1:0-86:14) to give the title compoundas a white powder (630 mg, 72%). ES-MS: calcd. for C₂₅H₃₀F₂N₄O₅(504.53). found: 505.4 [M+H].

1-{2-[(Benzyloxy-formyl-amino)-methyl]-hexanoyl}-4-trans-fluoro-pyrrolidine-2-carboxylicacid-(2-amino-5-fluoro-pyridin-N-oxide-2-yl)-amide

To a DCM solution of the compound (1.25 g, 2.56 mmol, 1 eq), MCPBA (1.32g, 7.68 mmom, 3 eq) was successfully added at 0° C. and the reaction wasstirred for 16 h. The reaction mixture is partitioned between NaHCO3 andthe DCM layer. The organic layer is dried over Na2SO4 and concentrated.The residue is purified by silica gel chromatography using DCM:Acetone(1:0-9:1) to yield the title compound (1.2 g

EXAMPLE 31-{2-[(Formyl-hydroxy-amino)-methyl]-hexanoyl}-pyrrolidine-2-carboxylicacid pyrazin-2-ylamide

The title compound is prepared according to General Procedure A from2-[(benzyloxy-formyl-amino)-methyl]-hexanoic acid A-7 (R=n-butyl) andpyrrolidine-2-carboxylic acid pyrazin-2-amide A-8 (X=CH₂, n=1,R₁=2-pyrazinyl).

EXAMPLE 41-{2-[(Formyl-hydroxy-amino)-methyl]-hexanoyl}-pyrrolidine-2-carboxylicacid pyridazin-3-ylamide

The title compound is prepared according to General Procedure A from2-{[formyl-(tetrahydro-pyran-2-yloxy)-amino]-methyl}-hexanoic acid A-7(R=n-butyl) and pyrrolidine-2-carboxylic acid pyridazin-3-amide A-8(X=CH₂, n=1, R₁=3-pyridazinyl).

Step 1: Pyridazin-3-ylamine

To a solution of 6-chloro-2-amino-pyridiazine (4 g) and NaOH (powdered,1.4 g) in ethanol (150 ml), 10% Pd/C (0.6 g) is added. The reactionmixture is stirred under Hydrogen atmosphere for 16 h. It is filteredthrough celite and the solvent was concentrated. The resulting residueis triturated with ether to provide the known amino compound.

Step 2: 2-(Pyridazin-3-ylcarbamoyl)-pyrrolidine-1-carboxylic acidtert-butyl ester

To a solution of Boc-Pro-OH (1 equiv) in DCM at 0° C., Ghosez Reagent(1.1 equiv) is added and the reaction mixture was stirred at 0° C. for 1h. To this the amine (1.1 equiv) in pyridine is added and the reactionmixture is stirred at room temperature for 16 h. It is then concentratedto remove all volatiles and redissolved in excess DCM. The organic layeris washed with 10% Citric acid, Brine and NaHCO₃, dried over Na2SO4 andconcentrated. The resulting residue is purified by flash chromatographyusing 10-40% Ethyl acetate in hexanes to provide the title compound.HPLC: YMC-Pak Pro C18, S-3 □m, 120 A, 50×4.6 mm I.D. Column; gradienteluent 0%-90% MeCN over 8.5 min, 1.5 mL/min; Retention time=4.14 min.

ES-MS: calcd. for C₁₄H₂₀N₄O₃ (292). found 293 [M+H].

Step 2: Pyrrolidine-2-carboxylic acid pyridazin-3-ylamide

HPLC: YMC-Pak Pro C18, S-3 □m, 120 A, 50×4.6 mm I.D. Column; gradienteluent 0%-90% MeCN over 8.5 min, 1.5 mL/min; Retention time=2.398 min.

ES-MS: calcd. for C₉H₁₂N₄O (192.1). found 193.2 [M+H].

Step 3:1-(2-{[Formyl-(tetrahydro-pyran-2-yloxy)-amino]-methyl}-hexanoyl)-pyrrolidine-2-carboxylicacid pyridazin-3-ylamide

The title compound is prerepared under HATU condition as described ingeneral procedure A.

HPLC: YMC-Pak Pro C18, S-3 □m, 120 A, 50×4.6 mm I.D. Column; gradienteluent 20%-90% MeCN over 8.5 min, 1.5 mL/min; Retention time=3.655 min.

ES-MS: calcd. for C₂₂H₃₃N₅O₅ (447). found 448 [M+H].

EXAMPLE 51-[2-Cyclopentylmethyl-3-(formyl-hydroxy-amino)-propionyl]-4-fluoro-pyrrolidine-2-carboxylicacid (5-fluoro-1-oxy-pyridin-2-yl)-amide

The title compound is prepared according to General Procedure A from2-cyclopentylmethyl-3-[formyl-(tetrahydro-pyran-2-yloxy)-amino]-propionicacid and pyrrolidine-2-carboxylic acid pyridin-2-ylamide A-8 (X=CHF,n=1, R₁=5-Fluoro 2-pyridyl).

1-{2-Cyclopentylmethyl-3-[formyl-(tetrahydro-pyran-2-yloxy)-amino]-propionyl}-4-fluoro-pyrrolidine-2-carboxylicacid (5-fluoro-1-oxy-pyridin-2-yl)-amide

EXAMPLE 61-[2-Cyclobutylmethyl-3-(formyl-hydroxy-amino)-propionyl]-pyrrolidine-2-carboxylicacid pyridazin-3-ylamide

The title compound is prepared according to General Procedure A from2-cyclobutylmethyl-3-[formyl-(tetrahydro-pyran-2-yloxy)-amino]-propionicacid A-7 (R=cyclobutylmethyl) and pyrrolidine-2-carboxylic acidpyridazin-2-amide A-8 (X=CH₂, n=1, R₁=3-pyridazinyl).

EXAMPLE 71-[2-Cyclobutylmethyl-3-(formyl-hydroxy-amino)-propionyl]-pyrrolidine-2-carboxylicacid pyrazin-2-ylamide

The title compound is prepared according to General Procedure A from2-cyclobutylmethyl-3-[formyl-(tetrahydro-pyran-2-yloxy)-amino]-propionicacid A-7 (R=cyclobutylmethyl) and pyrrolidine-2-carboxylic acidpyrazin-2-amide A-8 (X=CH₂, n=1, R₁=2-pyrazinyl).

EXAMPLE 81-[2-Cyclopentylmethyl-3-(formyl-hydroxy-amino)-propionyl]-pyrrolidine-2-carboxylicacid pyrazin-2-ylamide

The title compound is prepared according to General Procedure A from2-cyclopentylmethyl-3-[formyl-(tetrahydro-pyran-2-yloxy)-amino]-propionicacid A-7 (R=cyclopentylmethyl) and pyrrolidine-2-carboxylic acidpyrazin-2-amide A-8 (X=CH₂, n=1, R₁=2-pyrazinyl).

EXAMPLE 94-Fluoro-1-{2-[(formyl-hydroxy-amino)-methyl]-hexanoyl}-pyrrolidine-2-carboxylicacid pyrazin-2-ylamide

The title compound is prepared according to General Procedure A from2-[(benzyloxy-formyl-amino)-methyl]-hexanoic acid A-7 (R=n-butyl) andpyrrolidine-2-carboxylic acid pyrazin-2-amide A-8 (X=CHF, n=1,R₁=2-pyrazinyl).

EXAMPLE 101-[2-Cyclopentylmethyl-3-(formyl-hydroxy-amino)-propionyl]-4-fluoro-pyrrolidine-2-carboxylicacid pyrazin-2-ylamide

The title compound is prepared according to General Procedure A from2-cyclopentylmethyl-3-(formyl-hydroxy-amino)-propionic acid A-7(R=cyclopentyl methyl) and pyrrolidine-2-carboxylic acid pyrazin-2-amideA-8 (X=CHF, n=1, R₁=2-pyrazinyl).

EXAMPLE 111-[2-Cyclobutylmethyl-3-(formyl-hydroxy-amino)-propionyl]-4-fluoro-pyrrolidine-2-carboxylicacid pyrazin-2-ylamide

The title compound is prepared according to General Procedure A from2-cyclobutylmethyl-3-[formyl-(tetrahydro-pyran-2-yloxy)-amino]-propionicacid A-7 (R=cyclobutyl methyl) and pyrrolidine-2-carboxylic acidpyrazin-2-amide A-8 (X=CHF, n=1, R₁=2-pyrazinyl).

2-Cyclobutylmethyl-3-[formyl-(tetrahydro-pyran-2-yloxy)-amino]-propionicacid is prepared from 2-cyclobutylmethyl malonic acid as described forthe synthesis of the corresponding cyclopentylmethyl derivative inExample 1.

2-Cyclobutylmethyl-malonic acid

The title compound is prepared from (bromomethyl)cyclobutane

2-Cyclobutylmethyl-acrylic acid

4-Benzyl-3-(2-cyclobutylmethyl-acryloyl)-oxazolidin-2-one

4-Benzyl-3-[2-cyclobutylmethyl-3-(tetrahydro-pyran-2-yloxyamino)-propionyl]-oxazolidin-2-one

N-[3-(4-Benzyl-2-oxo-oxazolidin-3-yl)-2-cyclobutylmethyl-3-oxo-propyl]-N-(tetrahydropyran-2-yloxy)-formamide

2-Cyclobutylmethyl-3-[formyl-(tetrahydro-pyran-2 yloxy)-amino]-propionicacid

1-{2-Cyclobutylmethyl-3-[formyl-(tetrahydro-pyran-2-yloxy)-amino]-propionyl}-4-fluoro-pyrrolidine-2-carboxylicacid pyrazin-2-ylamide

EXAMPLE 121-[2-Cyclobutylmethyl-3-(formyl-hydroxy-amino)-propionyl]-4-fluoro-pyrrolidine-2-carboxylicacid pyrimidin-4-ylamide

The title compound is prepared according to General Procedure A from2-cyclobutylmethyl-3-[formyl-(tetrahydro-pyran-2-yloxy)-amino]-propionicacid A-7 (R=cyclobutyl methyl) and pyrrolidine-2-carboxylic acidpyrimidin-4-amide A-8 (X=CHF, n=1, R₁=2-pyrimidinyl).

EXAMPLE 131-[2-Cyclobutylmethyl-3-(formyl-hydroxy-amino)-propionyl]-4-fluoro-pyrrolidine-2-carboxylicacid pyridazin-3-ylamide

The title compound is prepared according to General Procedure A from2-cyclobutylmethyl-3-[formyl-(tetrahydro-pyran-2-yloxy)-amino]-propionicacid A-7 (R=cyclobutyl methyl) and pyrrolidine-2-carboxylic acidpyridazin-3-amide A-8 (X=CHF, n=1, R₁=3-pyridazinyl).

(2S,4R)-tert-butyl4-fluoro-2-(pyridazin-3-ylcarbamoyl)pyrrolidine-1-carboxylate

To a solution of Boc-Pro(F)—OH (5 g, 21.46 mmol, 1 equiv) in DCM at 0°C., Ghosez Reagent (3.1 ml, 23.61 mmol, 1.1 equiv) is added and thereaction mixture was stirred at 0° C. for 1 h. To this the amine (2.65g, 27.9 mmol, 1.3 equiv) in Pyridine is added at 0° C. and the reactionmixture is stirred at room temperature for 16 h. It is then concentratedto remove all volatiles and redissolved in excess DCM. The organic layeris washed with 10% Citric acid, NaCl(sat) and NaHCO₃(sat), dried overNa₂SO4 and concentrated. The resulting residue is purified by flashchromatography using 10-15% Acetone in Dichloromethane to provide thetitle compound.

(2S,4R)-4-fluoro-N-(pyridazin-3-yl)pyrrolidine-2-carboxamide

The Boc-protected amide is taken into 4M HCl/Dioxane and the reaction isstirred at room temperature for 5 h. The solvent are removed underreduced pressure and the residue is triturated with ether to give thetitle compounds(2S,4R)-1-((2R)-3-cyclobutyl-2-((N-(tetrahydro-2H-pyran-2-yloxy)formamido)methyl)propanoyl)-4-fluoro-N-(pyridazin-3-yl)pyrrolidine-2-carboxamide

To a cold DMF solution (15 mL) of the Versiacid, (500 mg, 1.77 mmol, 1equiv), DIEA (1.7 ml, 9.72 mmol, 5.5 equiv), Amine.HCl salt (550 mg,1.943 mmol, 1.1 equiv) and HATU (739 mg, 1.943 mmol, 1.2 equiv) areadded. The resulting reaction mixture is stirred for 16 h at roomtemperature. The mixture is partitioned between excess ethyl acetate and10% citric acid. The organic layer is washed with sat. NaCl and sat.NaHCO₃, dried over anhydrous Na₂SO₄, filtered, and concentrated. Theresidue is purified by silica gel chromatography using 10-25% Acetone inDCM to give the title compound (53%).

EXAMPLE 141-[2-Cyclobutylmethyl-3-(formyl-hydroxy-amino)-propionyl]-4-fluoro-pyrrolidine-2-carboxylicacid (2-oxy-pyridazin-3-yl)-amide

The title compound is prepared according to General Procedure A from2-Cyclobutylmethyl-3-[formyl-(tetrahydro-pyran-2-yloxy)-amino]-propionicacid A-7 (R=cyclobutyl methyl) and pyrrolidine-2-carboxylic acidpyridazin-1-oxo-3-amide A-8 (X=CHF, n=1, R₁=3-pyridazinyl N-oxide).

6-((2S,4R)-1-(tert-butoxycarbonyl)-4-fluoropyrrolidine-2-carboxamido)pyridazine1-oxide

To a solution of Boc-Pro(F)—OH (2.047 g, 8.154 mmol, 1 equiv) in DCM at0° C., Ghosez Reagent (1.2 ml, 8.97 mmol, 1.1 equiv) is added and thereaction mixture is stirred at 0° C. for 1 h. To this the amine (1.27 g,11.42 mmol, 1.4 equiv) in Pyridine is added at 0° C. and the reactionmixture is stirred at room temperature for 16 h. It is then concentratedto remove all volatiles and the residue is dissolved in excess DCM. Theorganic layer is washed with 10% Citric acid, NaCl (sat) and NaHCO₃(sat), dried over Na₂SO₄ and concentrated. The resulting residue ispurified by flash chromatography using 2-15% Acetone in Dichloromethaneto provide the title compound (61%).

6-((2S,4R)-4-fluoropyrrolidine-2-carboxamido)pyridazine 1-oxide

The Boc-protected amide is taken into 4M HCl/Dioxane and the reaction isstirred at room temperature for 5 h. All volatiles are removed and theresidue is triturated with ether to give the title compounds.

6-((2S,4R)-1-((2R)-3-cyclobutyl-2-((N-(tetrahydro-2H-pyran-2yloxy)formamido)methyl)propanoyl)-4-fluoropyrrolidine-2-carboxamido)pyridazine1-oxide

To a cold DMF solution (20 mL) of the Versiacid, (571 mg, 2 mmol, 1equiv), DIEA (2.51 ml, 14.4 mmol, 6 equiv), Amine.HCl salt (718 mg, 2.4mmol, 1.2 equiv) and HATU (913 mg, 2.4 mmol, 1.2 equiv) are added. Theresulting reaction mixture is stirred for 16 h at room temperature. Themixture is partitioned between excess ethyl acetate and 10% citric acid.The organic layer is washed with sat. NaCl and sat. NaHCO₃, dried overanhydrous Na₂SO₄, filtered, and concentrated. The residue is purified bysilica gel chromatography using 10-20% Acetone in DCM and then using2-8% methanol in DCM to give the title compound (44%). 1H NMR (DMSO-d₆):

EXAMPLE 151-[2-Cyclopentylmethyl-3-(formyl-hydroxy-amino)-propionyl]-4-fluoro-pyrrolidine-2-carboxylicacid pyridazin-3-ylamide

The title compound is prepared according to General Procedure A from2-Cyclopentylmethyl-3-[formyl-(tetrahydro-pyran-2-yloxy)-amino]-propionylacid A-7 (R=cyclopentyl methyl) and pyrrolidine-2-carboxylic acidpyridazin-2-amide A-8 (X=CHF, n=1, R₁=3-pyridazinyl).

(2S,4R)-1-((2R)-3-cyclopentyl-2-((N-(tetrahydro-2H-pyran-2-yloxy)formamido)methyl)propanoyl)-4-fluoro-N-(pyridazin-3-yl)pyrrolidine-2-carboxamide

EXAMPLE 161-[2-Cyclopentylmethyl-3-(formyl-hydroxy-amino)-propionyl]-4-fluoro-pyrrolidine-2-carboxylicacid (2-oxy-pyridazin-3-yl)-amide

The title compound is prepared according to General Procedure A from2-cyclopentylmethyl-3-[formyl-(tetrahydro-pyran-2-yloxy)-amino]-propionylacid A-7 (R=cyclopentyl methyl) and pyrrolidine-2-carboxylic acidpyridazin-2-amide A-8 (X=CHF, n=1, R₁=3-pyridazinyl N-oxide).

6-aminopyridazine 1-oxide

To a solution of 6-Aminopyridazine in acetone is added a solution ofMCPBA (1 equiv.) in acetone in one portion. The reaction mixture isallowed to stir at room temperature for 1 hour. The solvent is removedand ether is added to the residue. The solid is filtered and dried toyield the title compound. This is used as such in the next step.

6-((2S,4R)-1-(tert-butoxycarbonyl)-4-fluoropyrrolidine-2-carboxamido)pyridazine1-oxide

To a solution of Boc-Pro(F)—OH (2.047 g, 8.154 mmol, 1 equiv) in DCM at0° C., Ghosez Reagent (1.2 ml, 8.97 mmol, 1.1 equiv) is added and thereaction mixture is stirred at 0° C. for 1 h. To this the amine (1.27 g,11.42 mmol, 1.4 equiv) in Pyridine is added at 0° C. and the reactionmixture is stirred at room temperature for 16 h. It is then concentratedto remove all volatiles and the residue is dissolved in excess DCM. Theorganic layer is washed with 10% Citric acid, NaCl(sat) and NaHCO₃(sat),dried over Na₂SO₄ and concentrated. The resulting residue is purified byflash chromatography using 2-15% Acetone in Dichloromethane to providethe title compound (61%).

6-((2S,4R)-4-fluoropyrrolidine-2-carboxamido)pyridazine 1-oxide

The Boc-protected amide was taken into 4M HCl/Dioxane and the reactionwas stirred at room temperature for 5 h. All volatiles were removed andthe residue was triturated with ether to give the title compounds.

6-((2S,4R)-1-((2R)-3-cyclopentyl-2-((N-(tetrahydro-2H-pyran-2-yloxy)formamido)methyl)propanoyl)-4-fluoropyrrolidine-2-carboxamido)pyridazine1-oxide

EXAMPLE 171-[2-Cyclohexylmethyl-3-(formyl-hydroxy-amino)-propionyl]-4-fluoro-pyrrolidine-2-carboxylicacid pyridazin-3-ylamide

The title compound is prepared according to General Procedure A from2-Cyclohexylmethyl-3-[formyl-(tetrahydro-pyran-2-yloxy)-amino]-propionylacid A-7 (R-cyclohexyl methyl) and pyrrolidine-2-carboxylic acidpyridazin-3-amide A-8 (X=CHF, n=1, R₁=3-pyridazinyl).

2-Cyclohexylmethyl-3-(formyl-hydroxy-amino)-propionic acid buildingblock is prepared from 2-cyclohexylmethylmalonic acid as described forthe synthesis of the corresponding cyclohexylmethylmalonic acid inExample 1.

2-Cyclohexylmethyl-malonic acid

The title compound is prepared from (bromomethyl)cyclohexane.

2-Cyclohexylmethyl-acrylic acid

4-Benzyl-3-(2-cyclohexylmethyl-acryloyl)-oxazolidin-2-one

4-Benzyl-3-[2-cyclohexylmethyl-3-(tetrahydro-pyran-2-yloxyamino)-propionyl]-oxazolidin-2-one)

N-[3-(4-Benzyl-2-oxo-oxazolidin-3-yl)-2-cyclohexylmethyl-3-oxo-propyl]-N-(tetrahydropyran-2-yloxy)-formamide

EXAMPLE 181-{4-Cyclopropyl-2-[(formyl-hydroxy-amino)-methyl]-butyryl}-4-fluoro-pyrrolidine-2-carboxylicacid pyrazin-2-ylamide

The title compound is prepared according to General Procedure A from2-cyclopropylethyl-3-[formyl-(tetrahydro-pyran-2-yloxy)-amino]-propionicacid A-7 (R=cyclopropyl ethyl) and pyrrolidine-2-carboxylic acidpyrazin-2-amide A-8 (X=CHF, n=1, R₁=2-pyrazinyl).

2-Cyclopropylethyl-3-(formyl-hydroxy-amino)-propionic acid buildingblock is prepared from 2-cyclopropylethylmalonic acid as described forthe synthesis of the corresponding cyclopentylmethylmethyl malonic acidin Example 1.

(Bromoethyl)cyclopropane

The title compound is prepared from 2-cyclopropylethanol.

2-Cyclopropylethyl-malonic acid

2-Cyclopropylethyl-acrylic acid

4-Benzyl-3-(2-cyclopropylethyl-acryloyl)-oxazolidin-2-one

4-Benzyl-3-[2-cyclopropylethyl-3-(tetrahydro-pyran-2-yloxyamino)-propionyl]-oxazolidin-2-one

N-[3-(4-Benzyl-2-oxo-oxazolidin-3-yl)-2-cyclopropylethyl-3-oxo-propyl]-N-(tetrahydropyran-2-yloxy)-formamide

2-Cyclopropylethyl-3-[formyl-(tetrahydro-pyran-2-yloxy)-amino]-propionicacid

1-{2-Cyclopropylethyl-3-[formyl-(tetrahydro-pyran-2-yloxy)-amino]-propionyl}-4-fluoro-pyrrolidine-2-carboxylicacid pyrazin-2-ylamide

EXAMPLE 19 Inhibition of Peptide Deformylase Activity

A PDF/FDH coupled assay (Lazennec et al., Anal. Biochem., Vol. 224, pp.180-182 (1997)) is used. In this coupled assay, the formate released byPDF from its substrate fMAS is oxidized by the coupling enzyme FDH,reducing one molecule of NAD⁺ to NADH, which causes an increase inabsorption at 340 nM. All assays are carried out at room temperature ina buffer of 50 mM HEPES, pH 7.2, 10 mM NaCl, 0.2 mg/mL BSA, in half-area96-well microtiter plates (Corning). The reaction is initiated by addinga mixture of 0.5 Unit/mL FDH, 1 mM NAD⁺, and fMAS at the desiredconcentration. To determine IC₅₀ (the concentration needed to inhibit50% of enzyme activity) values, PDF is pre-incubated for 10 minutes withvarying concentrations of the inhibitor, and the deformylation reactionis initiated by the addition of reaction mixture containing 4 mM fMAS.The initial reaction velocity, y, is measured as the initial rate ofabsorption increase at 340 nM using a SpectraMax plate reader (MolecularDevices, Sunnyvale, Calif.). The inhibitor concentration [ln] at which50% of the enzyme activity is inhibited, IC₅₀, is calculated using thefollowing formula:

y=y _(o)/(1+[ln]/IC₅₀)

where y_(o) is the reaction velocity in the absence of inhibitor.Solving this equation for IC₅₀ at the [ln] when y=y_(o)/2 yields IC₅₀.The IC₅₀ is calculated based on a nonlinear least-square regression fitusing a commercial software package (Deltapoint, Inc., Chicago, Ill.).

Using this assay, the IC₅₀ of various compounds are determined. The IC₅₀for the various compounds is determined against deformylase enzymecontaining nickel and zinc as the metal ion. The IC₅₀ values ofpreferred compounds of formula (I) determined for the zinc-containingdeformylase range from about 0.001 μM to about 0.2 μM. The IC₅₀ valuesof preferred compounds of formula (I) determined for thenickel-containing deformylase range from about 0.005 μM to about 3 μM.

EXAMPLE 20 Assay for Testing Antimicrobial Activity

Minimum inhibitory concentrations (MICs) are determined using themicrodilution method in 96-well format plates. Compounds are suspendedin DMSO at 5 or 10 mg/mL and stored at 4° C. until used. They arediluted in Mueller-Hinton Broth (MHB) or Trypticase Soy Broth (TSB) andused for MIC determination. The range of concentrations tested is64-0.0625 μg/mL final concentration using a two-fold dilution system.

The inoculum is prepared from cells grown on Trypticase Soy Agar (TSA)and incubated overnight at 35° C., 5-10 colonies are used to inoculateMHB or TSB broths, and the culture is incubated overnight at 35° C. Theovernight culture is diluted 1:10, incubated for 1 hour at 35° C.,diluted to the appropriate inoculum size and applied to the wellscontaining broth and test compound. Inoculum sizes are 2×10⁴ CFU/mL.

Plates are incubated at 35° C. for 48 hours and MIC are recorded after18 hours of incubation for bacteria. MIC is defined as the lowestconcentration of compound that does not produce visible growth afterincubation.

Minimum inhibitory concentration for various preferred compounds offormula (I) ranges from about 0.25 μg/mL to about 32 μg/mL against H.influenza (four strains), from about 0.001 μg/mL to greater than 8 μg/mLagainst S. aureus (four strains), from about 0.016 μg/mL to about 16μg/mL against S. pneumonia (four strains), and from about 0.008 μg/mL toabout 16 μg/mL against M. catarrhalis. The deformylase enzyme isobtained from E. coli.

The following are representative pharmaceutical formulations containinga compound of formula (I).

EXAMPLE 21 Tablet Formulation

The following ingredients are mixed intimately and pressed into singlescored tablets:

Quantity per Ingredient Tablet (mg) Compound of this invention 400Cornstarch 50 Croscarmellose sodium 25 Lactose 120 Magnesium stearate 5

EXAMPLE 22 Capsule Formulation

The following ingredients are mixed intimately and loaded into ahard-shell gelatin capsule:

Quantity per Ingredient Capsule (mg) Compound of this invention 200Lactose, spray -- dried 148 Magnesium stearate 2

EXAMPLE 23 Suspension Formulation

The following ingredients are mixed to form a suspension for oraladministration:

Ingredient Amount Compound of this invention 1.0 g Fumaric acid 0.5 gSodium chloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 gGranulated sugar 25.0 g Sorbitol (70% solution) 13.00 g Veegum K(Vanderbilt Co.) 1.0 g Flavoring 0.035 mL Colorings 0.5 mg Distilledwater q.s. to 100 mL

EXAMPLE 24 Injectable Formulation

The following ingredients are mixed to form an injectable formulation:

Ingredient Amount Compound of this invention 0.2-20 mg Sodium acetatebuffer solution, 0.4 M 20 mL HCl (1 N) or NaOH (1 N) q.s. to suitable pHWater (distilled, sterile) q.s. to 20 mL

EXAMPLE 25 Suppository Formulation

A suppository of total weight 2.5 g is prepared by mixing the compoundof the invention with Witepsol® H-5 (triglycerides of saturatedvegetable fatty acid; Riches-Nelson, Inc., New York), and has thefollowing composition:

Compound of the invention 500 mg Witepsol ® H-15 Balance

1. A compound of formula (I)

wherein R1 is hydrogen, alkyl, heteroalkyl, heterocycloalkyl, aryl orheteroalkyl; R3 is hydrogen, halogen, or alkoxy; R4 is aryl, orheteroaryl; or n is 0 to 3 and provided that R1 is cycloalkyl and/or R4is an optionally substituted 6 membered, monocyclic heteroaryl ringhaving 2, 3 or 4 nitrogen heteroatoms in the ring wherein one or more ofthe ring nitrogen heteroatoms is optionally oxidized or a salt thereofor a prodrug thereof.
 2. A compound according to claim 1 wherein R3 isselected from fluorine, chlorine or iodine.
 3. A compound according toclaim 1 wherein R1 is cycloalkyl.
 4. A compound according to claim 1wherein R4 is a heteroaryl of formula (II)

wherein each of R6, R7, R8 and R9 independently is hydrogen, alkyl,substituted alkyl, phenyl, halogen, hydroxyl, alkoxy or a salt thereofor a prodrug thereof.
 5. A compound according to claim 1 wherein R4 is aheteroaryl of formula (II.1) and

wherein R6, R7 and R8 are hydrogen and R8 is fluoro, a salt thereof or aprodrug thereof.
 6. A compound according to claim 1 wherein R4 is aheteroaryl of formula (II.2) and

wherein R5, R7 and R8 are hydrogen, a salt thereof or a prodrug thereof.7. A compound according to claim 1 wherein R4 is a heteroaryl of formula(II.3) and

wherein R6, R7 and R8 are hydrogen, a salt thereof or a prodrug thereof.8. A compound according to claim 1 wherein R4 is a heteroaryl of formula(II.4) and

wherein R6, R7 and R8 are hydrogen, a salt thereof or a prodrug thereof.8. (canceled)
 9. A compound according to claim 1 wherein R1 is selectedfrom cyclohexyl, cyclopentyl, cyclobutyl or cyclopropyl.
 10. A compoundaccording to claim 1 wherein R1 is an n-butyl.
 11. A compound accordingto claim 9 wherein R4 is selected from 2-amino, 5-fluoropyridineN-oxide, 2-pyrazine, 3-pyridazine, 3-pyrimidine, 4-pyrimidine or3-pyridazine N-oxide.
 12. A compound according to claim 10 wherein R4 isselected from 2-amino, 5-fluoropyridine N-oxide, 2-pyrazine,3-pyridazine, 3-pyrimidine, 4-pyrimidine or 3-pyridazine N-oxide.
 13. Acompound according to claim 9 wherein R3 is fluorine.
 14. A compoundaccording to claim 10 wherein R3 is fluorine.
 15. A compound accordingto claim 1 wherein R4 is a heteroaryl of the formula (II.5)

wherein R6, R7 and R8 are hydrogen, a salt thereof or a prodrug thereof.